Molecular and Cellular Biochemistry

, Volume 400, Issue 1–2, pp 9–15 | Cite as

The fused anthranilate synthase from Streptomyces venezuelae functions as a monomer

  • Meseret Ashenafi
  • Prasad T. Reddy
  • James F. Parsons
  • W. Malcolm Byrnes


Recently, we showed that the fused chorismate-utilizing enzyme from the antibiotic-producing soil bacterium Streptomyces venezuelae is an anthranilate synthase (designated SvAS), not a 2-amino-2-deoxyisochorismate (ADIC) synthase, as was predicted based on its amino acid sequence similarity to the phenazine biosynthetic enzyme PhzE (an ADIC synthase). Here, we report the characterization of SvAS using steady-state kinetics, gel filtration chromatography, and laser light scattering. The recombinant His-tagged enzyme has Michaelis constants Km with respect to substrates chorismate and glutamine of 8.2 ± 0.2 μM and 0.84 ± 0.05 mM, respectively, and a catalytic rate constant k cat of 0.57 ± 0.02 s−1 at 30 °C. Unlike most other anthranilate synthases, SvAS does not utilize ammonia as a substrate. The enzyme is competitively but non-cooperatively inhibited by tryptophan (K i = 11.1 ± 0.1 μM) and is active as a monomer. The finding that SvAS is a monomer jibes with the variety of association modes that have been observed for anthranilate synthases from different microorganisms, and it identifies the enzyme’s minimal functional unit as a single TrpE–TrpG pair.


Anthranilate synthase Chorismate-utilizing enzyme Fused enzyme Streptomyces venezuelae 



We acknowledge support from the U. S. National Institutes of Health (NIH) RCMI (Grant number 2G12RR003048-18) and MBRS-SCORE programs (Grants number 3S06GM0816-33S1 and 1SC3GM083752) to WMB. We thank Tin-Wein Yu, formerly of the University of Washington, for assistance with the development of protocols for expression of SvAS, Santiago Ramon-Maiques of the Centro Nacional de Investigaciones Oncológicas in Madrid for helpful discussions about the structure of the enzyme, and Michael J. Eck of Harvard University for providing us with the high resolution images used in Fig. 4. Disclaimer: certain commercial equipment, instruments, and materials are identified in this paper in order to specify the experimental procedure as completely as possible. In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the material, instrument, or equipment identified is necessarily the best available for the purpose.


  1. 1.
    Kane JF, Jensen RA (1970) The molecular aggregation of anthranilate synthase in Bacillus subtilis. Biochem Biophys Res Commun 41:328–333CrossRefPubMedGoogle Scholar
  2. 2.
    Patel N, Holmes WM, Kane JF (1974) Homologous and hybrid complexes of anthranilate synthase from Bacillus species. J Bacteriol 119:220–227PubMedCentralPubMedGoogle Scholar
  3. 3.
    Sawula RV, Crawford IP (1973) Anthranilate synthetase of Acinetobacter calcoaceticus: separation and partial characterization of subunits. J Biol Chem 248:3573–3581PubMedGoogle Scholar
  4. 4.
    Queener SW, Queener SF, Meeks JR, Gunsalus IC (1973) Anthranilate synthase from Pseudomonas putida: purification and properties of a two-component enzyme. J Biol Chem 248:151–161PubMedGoogle Scholar
  5. 5.
    Patel N, Holmes WM, Kane JF (1973) Intergeneric complementation of anthranilate synthase subunits. J Bacteriol 114:600–602PubMedCentralPubMedGoogle Scholar
  6. 6.
    Kane JF, Holmes WM, Smiley KL, Jensen RA (1973) Rapid regulation of an anthranilate synthase aggregate by hysteresis. J Bacteriol 113:224–232PubMedCentralPubMedGoogle Scholar
  7. 7.
    De Troch P, Dosselaere F, Keijers V, de Wilde P, Vanderleyden J (1997) Isolation and characterization of the Azospirillum brasilense trpE(G) gene, encoding anthranilate synthase. Curr Microbiol 34:27–32CrossRefPubMedGoogle Scholar
  8. 8.
    Bae YM, Holmgren E, Crawford IP (1989) Rhizobium meliloti anthranilate synthase gene: cloning, sequence, and expression in Escherichia coli. J Bacteriol 171:3471–3478PubMedCentralPubMedGoogle Scholar
  9. 9.
    Paradkar AS, Stuttard C, Vining LC (1991) Molecular cloning of the genes for anthranilate synthetase from Streptomyces venezuelae ISP 5230. FEMS Microbiol Lett 62:177–181CrossRefPubMedGoogle Scholar
  10. 10.
    Baker TI, Crawford IP (1969) Anthranilate synthase: partial purification and some kinetic studies on the enzyme from Escherichia coli. J Biol Chem 241:5577–5584Google Scholar
  11. 11.
    Tamir H, Srinivasan PR (1969) Purification and properties of anthranilate synthase from Salmonella typhimurium. J Biol Chem 244:6507–6513PubMedGoogle Scholar
  12. 12.
    Henderson EJ, Nagano H, Zalkin H, Hwang LH (1970) The anthranilate synthetase-anthranilate 5-phosphoribosyltransferase complex of Salmonella typhimurium. Implications concerning the mode of assembly of the complex. Biochemistry 13:1416–1423Google Scholar
  13. 13.
    Bauerle R, Hess J, French S (1987) Anthranilate synthase-anthranilate phosphoribosyltransferase complex and subunits of Salmonella typhimurium. Methods Enzymol 142:366–386CrossRefPubMedGoogle Scholar
  14. 14.
    Zalkin H, Hwang LH (1971) Anthranilate synthetase from Serratia marcescens: on the properties and relationship to the enzyme from Salmonella typhimurium. J Biol Chem 246:6899–6907Google Scholar
  15. 15.
    Robb F, Hutchinson MA, Belser WL (1971) Anthranilate synthetase: some physical and kinetic properties of the enzyme from Serratia marcescens. J Biol Chem 246:6908–6912PubMedGoogle Scholar
  16. 16.
    Tutino ML, Tosco A, Marino G, Sannia G (1997) Expression of Sulfolobus solfataricus trpE and trpG genes in E. coli. Biochem Biophys Res Commun 230:306–310CrossRefPubMedGoogle Scholar
  17. 17.
    Byrnes WM, Vilker VL (2004) Extrinsic factors potassium chloride and glycerol induce thermostability in recombinant anthranilate synthase from Archaeoglobus fulgidus. Extremophiles 8:455–462CrossRefPubMedGoogle Scholar
  18. 18.
    Morollo AA, Eck MJ (2001) Structure of the cooperative allosteric anthranilate synthase from Salmonella typhimurium. Nature Struct Biol 8:243–247CrossRefPubMedGoogle Scholar
  19. 19.
    Spraggon G, Kim C, Nguyen-Huu X, Yee M-C, Yanofsky C, Mills SE (2001) The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates, chorismate and glutamine, and a product, glutamate, and (ii) its end-product inhibitor, l-tryptophan. Proc Natl Acad Sci USA 98:6021–6026PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Knöchel T, Ivens A, Hester G, Gonzalez A, Bauerle R, Wilmanns M, Kirschner K, Jansonius JN (1999) The crystal structure of anthranilate synthase from Sulfolobus solfataricus: functional implications. Proc Natl Acad Sci USA 96:9479–9484PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Zalkin H, Kling D (1968) Anthranilate synthetase. Purification and properties of component I from Salmonella typhimurium. Biochemistry 7:3566–3573CrossRefPubMedGoogle Scholar
  22. 22.
    Ashenafi M, Carrington R, Collins AC, Byrnes WM (2008) The fused TrpEG from Streptomyces venezuelae is an anthranilate synthase, not a 2-amino-2-deoxyisochorismate (ADIC) synthase. Ethn Dis 18(S2):9–13Google Scholar
  23. 23.
    Lin C, Paradkar AS, Vining LC (1998) Regulation of an anthranilate synthase gene in Streptomyces venezuelae by a trp attenuator. Microbiol 144:1971–1980CrossRefGoogle Scholar
  24. 24.
    Grisostomi C, Kast P, Pulido R, Huynh J, Hilvert D (1997) Efficient in vivo synthesis and rapid purification of chorismic acid using an engineered Escherichia coli strain. Bioorg Chem 25:297–305CrossRefGoogle Scholar
  25. 25.
    Addadi L, Jaffe EK, Knowles JR (1983) Secondary tritium isotope effects as probes of the enzymic and nonenzymic conversion of chorismate to prephenate. Biochemistry 22:4494–4501CrossRefPubMedGoogle Scholar
  26. 26.
    Caligiuri MG, Bauerle R (1991) Subunit communication in the anthranilate synthase complex from Salmonella typhimurium. Science 252:1845–1848CrossRefPubMedGoogle Scholar
  27. 27.
    Li QA, Mavrodi DV, Thomashow LS, Roessle M, Blankenfeldt W (2011) Ligand binding induces an ammonia channel in 2-amino-2-desoxyisochorismate (ADIC) synthase PhzE. J Biol Chem 286:18213–18221PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Ziebart KT, Dixon SM, Avila B, El-Badri MH, Guggenheim KG, Kurth MJ, Toney MD (2010) Targeting multiple chorismate-utilizing enzymes with a single inhibitor: validation of a three-stage design. J Med Chem 53:3718–3729CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Meseret Ashenafi
    • 1
  • Prasad T. Reddy
    • 2
    • 3
  • James F. Parsons
    • 3
  • W. Malcolm Byrnes
    • 1
  1. 1.Department of Biochemistry and Molecular Biology, College of MedicineHoward UniversityWashingtonUSA
  2. 2.Biomolecular Measurement DivisionNational Institute of Standards and TechnologyGaithersburgUSA
  3. 3.University of Maryland, Institute for Bioscience and Biotechnology ResearchRockvilleUSA

Personalised recommendations